1. Field of the Invention
The present invention relates to compounds and compositions for interfering with the association of Myc and Max. These compounds and compositions are useful in methods for inhibiting growth or proliferation of a cell. Methods of inhibiting growth or proliferation of a cell comprise contacting the cell with an amount of a compound that interferes with Myc and Max association effective to inhibit growth or proliferation of the cell. The compounds exhibit increased inhibitory activity against c-Myc relative to the known c-Myc inhibitor small-molecule N-([1,1′-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (10074-G5).
2. Description of the Related Art
c-Myc is important in the transcription of a myriad of genes involved in roles that include cell proliferation, apoptosis, differentiation and metabolism.(1-6) Though Myc is essential for normal cell function, its over-expression and/or dys-regulation is associated with a wide variety of human cancers, including, but not limited to, leukaemias and Burkitt's lymphoma, as well as cancers of the breast, pancreatic, lung and colon. (8-14, 20-21) Given its central role in many key cellular processes, there is concern that, by definition, c-Myc must possess a narrow therapeutic window, which would render the development of c-Myc inhibitors that are non-toxic to normal cells especially challenging. (15) However, recently there have been several reports that the inhibition of c-Myc, whilst lethal to transformed cells, exhibits only mild and reversible effects on normal cells.(8,16,17) Moreover, it now appears that c-Myc actually exerts an amplifying control on transcription of its target genes,(18,19) such that its inhibition would not result in complete inactivation of transcription. Together, these findings support the idea that the inhibition of c-Myc is a viable strategy towards the development of targeted antineoplastics.
In 2012, it was estimated there would be about 577,190 cancer deaths in the United States alone (39), about one-seventh of which will be associated with tumours exhibiting changes in the c-myc proto-oncogene or its expression (1). Inactivation of Myc results in cell-cycle arrest, apoptosis and tumour regression (40-42). Thus, the inhibition of Myc function is an appealing tactic to expand the arsenal of anticancer therapeutics in a highly targeted manner. Currently, there is no such “Myc drug” in the clinic, and the most potent Myc inhibitors to date exhibit only low micromolar IC50 values (20). Given the significant role of Myc in the development and progression of a wide variety of cancers, there is an urgent need for more potent and diverse Myc inhibitors.
c-Myc (Myc) is a short-lived (t1/2=20-30 min) nuclear oncoprotein and is a member of the basic-helix-loop-helix leucine zipper (bHLH-LZ) protein family of transcription factors that includes its obligate binding partner, Max, and its antagonist proteins of the Mad family (1). In its monomeric form, c-Myc is intrinsically disordered and transcriptionally inactive. However, upon dimerization with the bHLH-ZIP relative Max, an obligatory event that is required for all of c-Myc's known biological activities,(1-5) c-Myc and Max assume a coiled coil structure that recognizes the E-box sequence (5′-CACGTG-3′) in DNA.(6) Subsequently, various co-activators are recruited to initiate transcription. Unlike c-Myc, Max is capable of forming homodimers, as well as heterodimers with other bHLH-ZIP family members that include Mad, Mxi-1 (Mad2) and Mnt.(7) Although they repress transcription, Max-Max, Max-Mad and Max-Mxi-1 dimers retain the ability to bind E-box elements, and, therefore, compete with c-Myc-Max heterodimers, which provides a means by which c-Myc's transcriptional activity is kept in check.(7)
In its monomeric form, the bHLH-LZ domain of Myc (and Max) is intrinsically disordered, presenting no obvious binding sites for the development of inhibitors (6). Myc becomes transcriptionally functional only upon its heterodimerization with Max, an event in which the two proteins act as each other's chaperone to generate a parallel, left-handed four-helix bundle structure that recognizes the obligate E-box DNA sequence CAC/TGTG. The Myc-Max heterodimer thus serves as an excellent example of the coupled folding and binding of two intrinsically disorderd proteins. Indeed, most biological activities of Myc require this interaction (43-45). The basic regions of Myc and Max bind the DNA, while the HLH and LZ domains form the dimerization interface. Unlike Myc, Max is constitutively expressed, is stable (t1/2>24 h) and can also homodimerize. Max-Max homodimers bind the same DNA sequence as Myc-Max heterodimers but rather inhibit transcription, which is manifested through the direct competition for Max and for the common DNA site.
Since the dimerization of c-Myc and Max is an essential criterion for c-Myc to become functional, it follows that the disruption of the protein-protein interactions between c-Myc and Max should be an effective approach towards the inhibition of c-Myc's transcriptional activity. However, monomeric c-Myc's intrinsic disorder renders rational drug design a significant challenge, with the protein bereft of any recognizable sites that molecules could dock into or that computationally assisted docking programs could enlist as potential binding sites in virtual screens. (20,21) Furthermore, there does not appear to be any obvious binding pockets on the structure c-Myc-Max heterodimer, either. Unsurprisingly, therefore, the majority of small-molecule inhibitors of c-Myc-Max dimerization have been identified through screening large chemical libraries, and span structurally diverse chemotypes.(20) These small-molecules include rhodanine and thiazolidine-2,5-dione derivatives,(22,23) peptidomimetic-based inhibitors,(24,25) “credit-card” compounds that are based on planar, hydrophobic scaffolds designed to “slot” into the c-Myc-Max leucine zipper,(26) and the pyrazolo[1,5-a]pyrimidine “Mycro” compounds,(27, 28) as well as indirect c-Myc inhibitors, such as “JQ-1”, a thieno-triazolobenzo-1,4-diazepine that inhibits bromodomain-containing proteins. (29) A select few of these have been characterized more fully and shown to operate through the recognition and stabilization of monomeric, transcriptionally inactive c-Myc, preventing it from undergoing its obligatory dimerization with Max.(30,31) Two such compounds are 10058-F4 ((Z)-5-(4-ethylbenzylidene)thiazolidine-2,4-dione) and 10074-G5 (N-([1,1′-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine), the former of which was previously developed into more potent congeners.(23)
Owing to a lack of recognizable binding clefts or motifs, coupled with an inability to predict the structures of potential Myc-Max disruptors, the majority of inhibitors reported to date have been identified by screening large libraries of compounds, as stated above, and most compounds identified to date have been hampered by low affinity binding and/or poor in vivo activity against Myc-dependent tumors (21, 35, 49). Accordingly, there is a need for new compounds that inhibit Myc-activity.